Isolation of lectins

ABSTRACT

The invention relates to a process for isolating a lectin composition, in particular comprising the mannose-binding lectin (MBL), suitable for using recombinantly produced lectins as starting material, as well as methods for enriching a lectin preparation with respect to high molecular weight lectins. The method implies the use of sugar derivative modified solid supports, wherein the concentration of sugar derivatives on the solid support contributes to the isolation of predetermined oligomers of lectins.

[0001] The invention relates to a process for isolating a lectincomposition, in particular comprising the mannose-binding lectin (MBL),suitable for using recombinantly produced lectins as starting material,as well as methods for enriching a lectin preparation with respect tohigh molecular weight lectins.

BACKGROUND OF THE INVENTION

[0002] Lectins are proteins characterized by the presence of acarbohydrate binding domain. Collectins are a group of lectinscomprising an oligomeric structure of subunits, each subunit having acarbohydrate binding domain. In vivo lectins are represented in avariety of number of subunits leading to populations of lectins withdifferent molecular mass.

[0003] MBL is a protein of the collectin family and characterized by anoligomeric structure of subunits each consisting of a calcium-dependent,C-type carbohydrate-recognition domain (CRD), attached to a collagenousrod. MBL activates the complement system via associated serine proteases(MASP—mannose-binding lectin associated serine proteases), i.e. by amechanism similar to C1q. Mannose-binding protein (MBL) is a protein tobe used for substitution or replacement therapy in patients withinherited or acquired MBL-deficiency associated with functional and/orclinical symptoms.

[0004] MBL derived from human blood plasma is assembled into an oligomerof subunits, each consisting of three identical polypeptide chains. Thenumber of subunits in an MBL molecule is varying [Lipscombe R J, et al:Distinct physicochemical characteristics of human mannose bindingprotein expressed by individuals of differing genotype, Immunology 85(1995) 660-667.], but it has been suggested that the biologically activepolypeptide is an oligomer consisting of more than three subunits.Plasma comprises oligomers of more than three subunits as well asdenatured and structurally impaired protein forms leading to bands onfor example SDS gels between the dominating MBL bands corresponding tothe higher oligomers.

[0005] Recombinantly produced MBL reveals an oligomer variation similarto plasma-derived MBL [Vorup-Jensen T et al: Recombinant expression ofhuman mannan-binding lectin, Int. Immunopharm. 1 (2001) 677-687].However, recombinantly produced MBL usually has a higher content oflow-mass forms than plasma derived MBL. Low-mass forms of MBL includefor example single polypeptide chains, single subunits, and dimericsubunits.

[0006] Lectins are typically isolated by applying a compositioncomprising the lectins to a column being modified with a sugar to whichthe lectins bind. However, the efficacy of the columns varies. In PCTapplication WO 00/70043 a method for separating high oligomers from lowmass forms is described, wherein recombinantly produced MBL is subjectedto fractionation on a special column, wherein the column as such doesnot have any affinity for the MBL.

DESCRIPTION OF THE INVENTION

[0007] A fast and simple method to isolate lectins, such asmannose-binding lectin (MBL), or derivatives and variants thereof(herein collectively designated lectins) from a solution is of majorimportance.

[0008] Moreover, to be in control of the mass distribution of lectins isalso of major importance, because different oligomers of said lectinpolypeptides possess different biological functions and activity.

[0009] The present invention describes a method to isolate lectins, suchas MBL, by applying the preparation to a solid support having coupledthereto a predetermined concentration of a sugar derivative. The methodcan be used as a unit operation during preparation, purification, and/orformulation of said polypeptide. Compared to other methods, it isadvantageous that the method of the invention can be applied to changethe composition of oligomers of said polypeptide, so that high-massoligomers of said polypeptide are separated from low-mass oligomers ofsaid polypeptide, or intermediate-mass oligomers are separated fromhigh-mass oligomers and low-mass oligomers.

[0010] Thus one objective of the present invention is to provide amethod for isolating at least one lectin from a preparation comprising avariety of lectin molecules, comprising

[0011] establishing a solid support having coupled thereto apredetermined concentration of a sugar derivative,

[0012] applying the preparation to said solid support,

[0013] allowing the lectins to bind to the sugar derivative coupled tothe solid support,

[0014] washing the solid support, with a washing liquid removing unboundcontaminants, and

[0015] optionally recovering said lectin(s) from the solid support.

[0016] By the term “predetermined concentration” is meant that apredetermined volume of sugar derivative having a predeterminedconcentration is applied to a predetermined volume or area of solidsupport allowing the sugar derivative to be coupled to the solidsupport. In one embodiment the term means that a predeterminedconcentration of sugar derivative is coupled to a predetermined volumeor area of the solid support.

[0017] By the term “contaminant” is meant contaminants in the lectinpreparation as well as lectins not being bound to the sugar derivativedue to their smaller molecular weight. In particular when desiringintermediate molecular weight lectins, the contaminants may comprise thedesired lectins when using two- or more step procedures as discussedbelow. Thus, the term “contaminant” is synonymous with unbound materialincluding unbound lectins.

[0018] By the term “a variety of lectin molecules” is preferably meant avariety of oligomers of lectin molecules, said lectin molecules havingidentical or substantially identical structural subunits, such asdescribed below.

[0019] It is a second objective of the present invention to provide suchsolid supports, having covalently coupled thereto a predeterminedconcentration of a sugar derivative, wherein the sugar derivative ispreferably an amino sugar.

[0020] In another aspect the invention relates to a method for changingthe oligomer distribution of lectins in solution. Hence, in oneembodiment the invention relates to a method for increasing the ratio Rof a composition comprising a variety of lectin molecules, wherein R isthe ratio of the concentration of lectin molecules having a highmolecular weight above a predetermined molecular weight to theconcentration of lectin molecules having a low molecular weight below orequal to the predetermined molecular weight, said method comprising

[0021] obtaining a lectin preparation comprising low molecular weightlectin and high molecular weight lectin, said preparation having theratio R=R₀,

[0022] applying the preparation to an isolation method, preferably themethod for isolating at least one lectin from a preparation describedherein above,

[0023] obtaining a composition comprising the recovered lectinmolecules.

[0024] Thereby a composition having an increased content of high massoligomers of lectins as compared to the starting material is obtained.

[0025] In particular the increase of the ratio leads to compositionscomprising lectins having a molecular weight above the molecular weightfor dimer lectins. Thus, in another aspect the invention relates to amethod for producing a composition comprising a variety of lectinmolecules, wherein substantially all of said lectin molecules have amolecular weight above a predetermined molecular weight for dimerlectins, said method comprising,

[0026] obtaining a lectin preparation comprising lectin molecules havinga high molecular weight above the predetermined molecular weight andlectin molecules having a low molecular weight below or equal to thepredetermined molecular weight, said preparation having the ratio R=R₀,wherein R is the ratio of the concentration of lectin molecules having ahigh molecular weight above the predetermined molecular weight to theconcentration of lectin molecules having a low molecular weight below orequal to the predetermined molecular weight,

[0027] applying the preparation to an isolation method, preferably themethod for isolating at least one lectin from a preparation describedherein above,

[0028] obtaining a composition comprising the recovered lectinmolecules.

[0029] Since the starting material for the methods comprises high masslectins as well as low mass lectins, the invention also relates to amethod for separating a composition comprising a variety of lectinmolecules, wherein substantially all of said lectin molecules have ahigh molecular weight above a predetermined molecular weight, from alectin preparation comprising lectin molecules having a high molecularweight above the predetermined molecular weight and lectin moleculeshaving a low molecular weight below or equal to the molecular weight fordimer lectins, said preparation having the ratio R=R₀, wherein R is theratio of the concentration of lectin molecules having a high molecularweight above the predetermined molecular weight to the concentration oflectin molecules having a low molecular weight below or equal to thepredetermined molecular weight, said method comprising

[0030] obtaining said lectin preparation,

[0031] applying the preparation for an isolation method, preferably themethod for isolating at least one lectin from a preparation describedherein above,

[0032] obtaining a composition comprising the recovered lectinmolecules.

[0033] For most purposes the high mass lectins are the desired ones;however, for some applications the low mass lectins are wanted, andaccordingly, the invention further relates to a method for producing acomposition comprising a variety of lectin molecules, whereinsubstantially all of said lectin molecules have a low molecular weightbelow or equal to a predetermined molecular weight, said methodcomprising,

[0034] obtaining a lectin preparation comprising lectin molecules havinga high molecular weight above the predetermined molecular weight andlectin molecules having a low molecular weight below or equal to thepredetermined molecular weight, said preparation having the ratio R=R₀,wherein R is the ratio of the concentration of lectin molecules having ahigh molecular weight above the predetermined molecular weight to theconcentration of lectin molecules having a low molecular weight below orequal to the predetermined molecular weight,

[0035] applying the preparation to an isolation method, preferably themethod for isolating at least one lectin from a preparation describedherein above,

[0036] obtaining a composition comprising the unbound lectin moleculesof the washing liquid.

[0037] For all the methods mentioned above, preferably the compositionof recovered lectin molecules has the ratio R=R₁, wherein R₁ is at least1, such as at least 1.05, such as at least 1.10, such as at least 1.15,such as at least 1.25, such as at least 1.50, such as at least 1.75,such as at least 2.0, such as at least 2.5, such as at least 3.0, suchas at least 4.0, such as at least 5.0, such as at least 6.0, such as atleast 7.0, such as at least 8.0, such as at least 9.0, such as at least10.0, such as at least 15, such as at least 20, such as at least 30,such as at least 40, such as at least 50, such as at least 60, such asat least 70, such as at least 80, such as at least 90, such as at least100, such as at least 100, such as at least 1000, such as at least10000.

[0038] For all of the methods disclosed above, the predeterminedmolecular weight is preferably the molecular weight for dimer lectins,more preferred for trimer lectins.

[0039] For most purposes high mass lectins are the desired ones;however, in certain embodiments intermediate mass lectins may be wanted.Accordingly, in a further aspect the present invention relates to amethod of increasing the ratio R of a composition comprising a varietyof lectin molecules, wherein R is the ratio of the concentration oflectins having an intermediate molecular mass to the concentration oflectin molecules having a high molecular mass and a low molecular mass,

[0040] wherein intermediate molecular mass is a molecular weight below afirst predetermined molecular weight and above a second predeterminedmolecular weight, high molecular mass is a molecular mass above or equalto the first predetermined molecular weight, and low molecular mass is amolecular mass below or equal to the second predetermined molecularweight,

[0041] said method comprising the steps of obtaining a lectin compostioncomprising low molecular mass lectin, intermediate molecular mass lectinand high molecular mass lectin, said preparation having the ratio R=R₀,

[0042] applying the composition to an isolation method, preferably themethod for isolating at least one lectin from a preparation describedherein above; and

[0043] obtaining a composition comprising the recovered lectinmolecules; and

[0044] optionally repeating the step of applying the composition to anisolation method, preferably the method for isolating at least onelectin from a preparation described herein above.

[0045] In one embodiment the first predetermined molecular weight may bethe molecular weight for heptamer lectins, and the second predeterminedmolecular weight is the molecular weight for dimer lectins.

[0046] In another aspect the invention relates to a method for isolatingat least one lectin from a preparation comprising a variety of lectinmolecules, comprising

[0047] establishing a solid support being derivatized with at least oneamino sugar,

[0048] applying the preparation to said solid support,

[0049] allowing the lectins to bind to the amino sugar coupled to thesolid support,

[0050] washing the solid support with a washing liquid removing unboundcontaminants, and

[0051] optionally recovering said lectin(s) from the solid support.

[0052] By the term “derivatized” is meant that the amino sugar iscoupled to the solid support as described above.

[0053] The lectin according to the invention may be any lectin whereinseveral oligomeric forms are produced, for example collectins, such asmannose-binding lectins. In particular the invention relates to a methodfor producing MBL compositions. By the term MBL is meant mannose-bindinglectin (or mannose-binding protein, as denoted by some authors). MBL ispreferably human MBL having a protein sequence as shown in for examplePCT application WO 00/70043 or derivatives or variants thereof beingfunctional equivalents of MBL. In the following the invention will bedescribed in relation to the lectin MBL.

[0054] The methods according to the present invention allow large scalemanufacturing of the lectins in question. Thus, in another aspect theinvention relates to any industrial or large scale method ofmanufacturing MBL comprising the application of said methods during themanufacturing of said MBL.

DRAWINGS

[0055]FIG. 1 illustrates the eluting profile of recombinantly producedMBL applied on and eluted from a column with immobilized D-mannosamineas described in example 1. The figure shows an SDS-PAGE Western usingmonoclonal antibodies raised against plasma derived MBL. MW=marker(kDa), A=Application (recombinantly derived MBL from before use of theinvention), R=Run Through fraction obtain during application; E=Elutingfraction after wash with EDTA (recombinantly derived MBL after use ofthe invention).

[0056]FIG. 2 illustrates the eluting profile of recombinantly producedMBL applied on and eluted from a column with immobilized D-mannosamineas described in example 2. The figure shows an SDS-PAGE Western usingmonoclonal antibodies raised against plasma derived MBL. ‘Dummy’: Columncoupled with no amino sugar, ‘Low-sub’=column coupled with low amount ofamino sugar, ‘High-sub’=column coupled with large amount of amino sugar,‘high-sub R’=column coupled with large amount of amino sugar, afterregeneration with NaOH. MW=marker (kDa), A=Application, R=Run Throughfraction obtain during application; E=Eluting fraction after wash withEDTA, Pl=Plasma derived MBL (control).

[0057]FIG. 3 illustrates the eluting profile of recombinantly producedMBL applied on and eluted from a column with immobilized D-glucosaminein two concentrations as described in example 5. The figure shows anSDS-PAGE Western using monoclonal antibodies raised against plasmaderived MBL. A=Application (recombinantly derived MBL from before use ofthe invention), R=Run Through fraction obtain during application;E=Eluting fraction after wash with EDTA (recombinantly derived MBL afteruse of the invention), Pl=Plasma derived MBL (control).

DETAILED DESCRIPTION OF THE INVENTION

[0058] Sugar Derivative

[0059] In a preferred embodiment the term sugar derivative means acompound derived from a sugar, wherein the derived compound possessesthe capability of being coupled to the solid support in a predeterminedoriented manner. The present inventors have identified one of thereasons why previous isolation methods may lack in efficiency. It hasthus been found that lectins bind to specific binding sites on thesaccharides, and when the prior art uses sugars coupled to column andother solid supports in a random manner, a percentage of the saccharidescoupled are not capable of binding the lectins since the binding sitesare not positioned in order to bind the lectin.

[0060] Thus, the sugar derivative according to the present inventionpreferably comprises a reactive group that allows oriented coupling tothe solid support. The sugar derivative may be any derivative capable ofbinding the lectin when the sugar derivative is coupled to the solidsupport. Sugar derivatives not capable of binding the lectin, when thederivative is in free form, i.e. un-coupled, may also be used accordingto the invention, when capable of binding the lectin in coupled form.

[0061] The predetermined orientation of the sugar may be obtained byincorporating a reactive group into the sugar, preferably at a specificposition, whereby it is possible to couple the sugar derivative to thesolid support in a specifically oriented manner, in that the couplingtakes place through the reactive group to the solid support. Thus, bythe term “oriented coupling” is meant that the sugar is coupled to thesolid support through a specific, predetermined group in the sugarmolecule, so that the free part of the sugar molecule is oriented in apredetermined position.

[0062] In a monosaccharide the binding site and the reactive group arepositioned in the same monosaccharide structure; however, indisaccharides and higher oligosaccharides and polysaccharides, thebinding site may be positioned in another monosaccharide structure thanthe reactive group. In the present context the term “monosaccharidestructure” means the structure capable of forming a ring in thesaccharide. For example, a disaccharide comprises two monosaccharidestructures. In a preferred embodiment the reactive group is positionedfar from the binding site thereby obtaining the best possibilities ofhaving a binding site not sterically hindered.

[0063] Sugars

[0064] The term sugar derivative means any compound being derived from asugar. In the present context sugar means any carbohydrate, includingmonosaccharides, disaccharides, trisaccharides, oligosaccharides, andpolysaccharides, whether being a five-membered ring (pentose) or asix-membered ring (hexose) or combinations thereof, or whether being aD-form or an L-form, as well as substances derived from monosaccharidesby reduction of the carbonyl group (alditols), by oxidation of terminalgroups to carboxylic acids, or by replacement of hydroxy groups byanother group. It also includes derivatives of these compounds.

[0065] Monosaccharides include polyhydroxy aldehydes H—[CHOH]_(n)—CHOand polyhydroxy ketones H—[CHOH]_(n)—CO—[CHOH]_(m)—H, i.e. aldoses,dialdoses, aldoketoses, ketoses, diketoses, deoxy sugars, amino sugars,and their derivatives.

[0066] Aldoses include monosaccharides with an aldehydic carbonyl, i.e.polyhydroxy aldehydes H—[CHOH]_(n)—CHO. Aldoses also includemonosaccharides with a potential aldehydic carbonyl group—a hemiacetalgroup—which arises from the ring closure of the aldose.

[0067] Ketoses include monosaccharides with a ketonic group, i.e.polyhydroxy ketones H—[CHOH]_(n)—CO—[CHOH]_(m)—H, i.e. ketones alsoinclude monosaccharides with a potential ketonic carbonyl group—ahemiketal group—which arises from the ring closure of the ketose.

[0068] Pyranoses are cyclic hemiacetals or cyclic hemiketals with atetrahydrofuran (five-membered ring).

[0069] Furanoses are cyclic hemiacetals or cyclic hemiketals with atetrahydrofuran (five-membered ring).

[0070] Dialdoses, diketoses, ketoaldoses (aldoketoses, aldosuloses),alditols are monosaccharides with more than one aldehyde and/or ketone.

[0071] Aldonic acids, ketonic acids are aldoses in which the aldehydegroup has been exchanged with a carboxy group.

[0072] Examples of derivatives of the sugars are uronic acids, aldoses,in which the first CH₂OH-group has been exchanged with a carboxy group;aldaric acids, aldonic acids, in which the first CH₂OH-group has beenexchanged with a carboxy group; deoxy sugars, monosaccharides, in whicha hydroxyl group has been exchanged with a hydrogen; amino sugars,monosaccharides, in which a hydroxyl group has been exchanged with anamino group.

[0073] Also glycosylamines are included, being amino sugars, in whichthe hydroxyl group on the hemiacetal group (formed after ring closure)has been exchanged with an amino group.

[0074] In the present context the term acetal is used in its normalmeaning, i.e. monosaccharides arising from ring closure of the aldose orketose. Furthermore, glycosides are acetals arising from elimination ofwater between the hemiacetal and hemiketal hydroxy group of amonosaccharide, oligosaccharide and polysaccharide, and the hydroxygroup of another monosaccharide, oligosaccharide and polysaccharide. Aglydosidic bond is the bond between the monosaccharides in a glycoside.

[0075] In the present context the term sugar, or any of the saccharidesand derivatives mentioned herein, includes all the various steric formsof the sugars, including the D-form and the L-form, α-form, and β-form.Thus, as an example, mannosamine means mannosamine in any of the stericforms, including D-mannosamine, L-mannosamine and (D,L)-mannosamine.Only when a specific steric form is used is this mentioned as forexample the D-mannosamine.

[0076] Specific Derivatives

[0077] The invention relates in particular to sugar derivatives having areactive group selected from amino groups, wherein the amino group mayfor example be selected from the group consisting of primary aminogroups, secondary amino groups and tertiary amino groups.

[0078] In particular the invention relates to the use of amino sugars asthe sugar derivative. The amino sugars may be amino monosaccharides,amino disaccharides, amino oligosaccharides, and amino polysaccharides,wherein at least one amino group is incorporated in a manner allowing itto act as a reactive group when coupling the amino sugar to the solidsupport.

[0079] Examples of suitable amino monosaccharides are:

[0080] Amino aldoses, such as mannosamine, glucosamine, allosamine,altrosamine, ribosamine, arabinosamine, gulosamine, idosamine,galactosamine, talosamine, xylosamine, lyxosamine. In particularD-mannosamine, D-glucosamine, D-allosamine, D-altrosamine, D-ribosamine,D-arabinosamine, L-gulosamine, L-idosamine, L-galactosamine,L-talosamine, L-xylosamine, L-lyxosamine.

[0081] Amino ketoses, such as sorbosamine, tagatosamine, psicosamine,fructosamine. In particular D-sorbosamine, D-tagatosamine,L-psicosamine, L-fructosamine.

[0082] Amino deoxyaldoses, such as derivatives of fucose, for examplefucosamine and fucosylamine, preferably L-fucosamine.

[0083] In particular in relation to isolation of MBL, the followingexamples of amino sugars are suitable: mannosamine, glucosamine,galactosamine, fructosamine. In particular D-mannosamine, D-glucosamine,L-galactosamine, L-fucosamine.

[0084] More preferably the amino sugars selected from the groupconsisting of glucosamine and mannosamine, most preferably amino sugarsselected from the group consisting of L-fucosamine, D-glucosamine andD-mannosamine may be used, in particular D-glucosamine andD-mannosamine. The formula of D-mannosamine is indicated herein below asfree D-mannosamine and as D-mannosamine bound to a resin.

[0085] Also disaccharides, oligosaccharides, and polysaccharides havingan amino group positioned as a reactive group in one part of themolecule, and a lectin binding site, in particular a MBL binding site inanother part of the molecule may be used.

[0086] For example the amino sugar may be any disaccharide derivativecomprising an amino group, wherein at least one of the monosaccharidesubunits of the disaccharide is selected from the group consisting ofmannose and glucose. Examples of preferred disaccharides includesaccharose derivatives, such as saccharosamine, and maltose derivatives,such as maltosamine, comprising an amino group, as well as agarosamine,cellulosamine, and amylosamine. A preferred disaccharide derivative issaccharosamine.

[0087] Furthermore the amino sugar may for example be a multimer ofglucosamine, such as a glucosamine dimer, a glucosamine oligomer or aglucosamine polymer.

[0088] Solid Support

[0089] The solid support may be any support used for capturing and/orisolating lectins. Thus the solid support may be any columns, filters orparticles used for isolation and purification, such as chromatographicresins, and magnetic particles.

[0090] Also, the solid support may be a surface capable of binding thelectin for example for further analyses, such as plastic surfaces andglass surfaces. Plastic surfaces may for example be selected from thegroup consisting of microtiter plates, ELISA wells and activatedmicrotiter plates. Activated microtiter plates may for example becyanuric chloride activated Covalink™ NH₂ Primary Amine from LifeTechnologies.

[0091] In one embodiment of the present invention, any solid supportcapable of covalently binding amines, preferably primary amines, may beused with the invention.

[0092] Useful columns may be any of the columns selected from the groupconsisting of N-N-hydroxy succinimide activated resins (for exampleNHS-Sepharose 4 FF from APBiotech, Affi-Gel 10 from BioRad, Affi-Gel 15from BioRad or Affi-Prep from BioRad), cyanogen bromide activated resins(for example CNBr-activated Sepharose 4 FF from APBiotech), ECHSepharose 4B from APBiotech, Activated CH-Sepharose 4B from AP-Biotechand epoxy activated resins (such as Epoxy-activated Sepharose 6B) aswell as their corresponding cross-linked resins.

[0093] A predetermined concentration of a sugar derivative may becoupled to the solid support according to the present invention. Thepredetermined concentration of the sugar derivative should be selectedaccording to what predetermined molecular weight is desirable for theparticular embodiment of the present invention.

[0094] The sugar derivative concentration is determined by two factors:

[0095] 1) concentration of positions (ligand arms) on the solid supportcapable of coupling a sugar derivative,

[0096] 2) concentration of sugar derivative per ligand arms.

[0097] The concentration of ligand arms per volume solid support ismostly determined by the production process of the solid support, andmay be controlled by coupling a labelled ligand to the solid support.

[0098] The concentration of sugar derivative per ligand arms isregulated by regulating the volume and concentration of sugar derivativeallowed to couple to the ligand arms.

[0099] In general a high predetermined concentration of sugar derivativeis used when a relatively low predetermined molecular weight isdesirable, and a low predetermined concentration of sugar derivative isused when a relatively high predetermined molecular weight is desirable.

[0100] The predetermined concentration of sugar derivative may forexample be determined by the amount of sugar derivative coupled to aspecific amount of solid support. For example, when the solid support isa chromatography column resin, the predetermined concentration ofmonosaccharide derivative is preferably from 0.1 mg sugar derivative perml resin to 1000 mg sugar derivative per ml resin, more preferablybetween 0.5 mg sugar derivative per ml resin and 500 mg sugar derivativeper ml resin, even more preferably from 2 mg sugar derivative per mlresin to 100 mg sugar derivative per ml resin.

[0101] In the present context a standard for the solid support is across-linked Sepharose resin modified with N-hydroxysuccinimide (NHS)residues, as described in the Examples. Preferably from 15-25 μmolligand arm per ml drained resin is used. Such a solid support isspecified to be able to bind 5 μmol dipeptide per ml drained resin, or1-2 μmol alfa-chymotrypsinogen per ml drained resin.

[0102] From this standardised solid support, intervals for thepredetermined concentration of sugar derivative to be coupled to theresin in order to obtain the lectins desired from the variety of lectinsare given.

[0103] Isolation of high molecular lectins as defined herein to be abovethe molecular weight for dimer lectins is obtained using the standardcolumn having coupled thereto a sugar derivative in a predeterminedconcentration of from 10 μmol per ml drained resin to 100 μmol per mldrained resin, such as from 20 μmol per ml drained resin to 80 μmol perml drained resin, such as from 30 μmol per ml drained resin to 70 μmolper ml drained resin, such as from 40 μmol per ml drained resin to 60μmol per ml drained resin. For monosaccharides said concentrationscorrespond to from 2 mg per ml drained resin to 20 mg per ml drainedresin, such as from 4 mg per ml drained resin to 16 mg per ml drainedresin, such as from 6 mg per ml drained resin to 14 mg per ml drainedresin, such as from 8 mg per ml drained resin to 12 mg per ml drainedresin.

[0104] Isolation of high molecular lectins as defined herein to be abovethe molecular weight for trimer lectins is obtained using the standardcolumn having coupled thereto a sugar derivative in a predeterminedconcentration of from 10 μmol per ml drained resin to 80 μmol per mldrained resin, such as from 20 μmol per ml drained resin to 70 μmol perml drained resin, such as from 30 μmol per ml drained resin to 70 μmolper ml drained resin, such as from 40 μmol per ml drained resin to 60μmol per ml drained resin.

[0105] Isolation of high molecular lectins as defined herein to be abovethe molecular weight for tetramer lectins is obtained using the standardcolumn having coupled thereto a sugar derivative in a predeterminedconcentration of from 10 μmol per ml drained resin to 60 μmol per mldrained resin, such as from 20 μmol per ml drained resin to 60 μmol perml drained resin, such as from 30 μmol per ml drained resin to 50 μmolper ml drained resin.

[0106] A high ligand solid support capable of binding also low molecularweight lectins, such as dimer lectins, is produced by using the standardcolumn having coupled thereto a sugar derivative in a predeterminedconcentration of above 200 μmol per ml drained resin, such as from above300 μmol per ml drained resin, such as from above 400 μmol per mldrained resin, such as from above 500 μmol per ml drained resin.

[0107] Washing Liquid

[0108] The washing liquid according to the present invention may be anysuitable washing liquid known to the person skilled in the art. Inparticular the buffer may comprise one or more components capable ofinterfering with the association between the lectin and the sugarderivative, such as the amino sugar.

[0109] In one embodiment of the present invention, the washing liquidcomprises one or more components selected from the group consisting ofsugars capable of binding to the lectin, sugar derivatives capable ofbinding to the lectin and divalent metal ion chelating agents.

[0110] Sugars and/or sugar derivatives capable of binding to the lectinmay for example be any monosaccharide and/or monosaccharide derivativecapable of binding to the lectin. For example any sugar derivativeuseful for coupling to the solid support according to the presentinvention (see herein above) or a corresponding non-derivatised sugar,or a corresponding sugar, which is further modified may be comprisedwithin the washing liquid.

[0111] Such monosaccharides may for example be selected from the groupconsisting of mannose, N-acetylmannosamine, glucose,N-acetylglucosamine, frucose, N-acetylfrucosamine, galactose andN-acetylgalactosamine. In particular, the monosaccharides may beselected from the group consisting of D-glucose, D-mannose, L-fucose andL-galactose.

[0112] Sugars and/or sugar derivatives capable of binding to the lectinmay also be any disaccharide, disaccharide derivative, trisaccharide,trisaccharide derivative, oligosaccharide, oligosaccharide derivative,polysaccharide or polysaccharide derivative wherein at least part thesugar is capable of binding to the lectin. Examples of usefuloligosaccharides include but are not limited to saccharose and maltose.

[0113] Divalent metal ion chelating agents are any agents capable ofchelating a divalent metal ion. The divalent metal ion may for examplebe Ca2+. Examples of divalent metal ion chelating agents include but arenot limited to EDTA and citrate.

[0114] Furthermore the washing liquid may be prepared in any othermanner suitable for elution of lectins known by the person skilled inthe art. For example the washing liquid may comprise a high saltconcentration or an acidic pH.

[0115] Lectins

[0116] The lectins isolated according to the present invention may beany lectins. For example the lectin may be a collectin, such as forexample MBL (mannose-binding lectin), SP-A (lung surfactant protein A),SP-D (lung surfactant protein D), BK (or BC, bovine conglutinin) andCL-43 (collectin-43). Collectins all exhibit the following architecture:they have an N-terminal cysteine-rich region that appears to forminter-chain disulfide bonds, followed by a collagen-like region, anα-helical coiled-coil region and finally a C-typ lectin domain which isthe pattern-recognizing region and is referred to as the carbohydraterecognition domain (CRD). The name collectin is derived from thepresence of both collagen and lectin domains. The α-helical coiled-coilregion initiates trimerisation of the individual polypetides to formcollagen triple coils, thereby generating collectin subunits eachconsisting of 3 individual polypeptides, whereas the N-terminal regionmediates formation of oligomers of subunits. Different collectinsexhibit distinctive higher order structures, typically either tetramersof subunits or hexamers of subunits.

[0117] In a preferred embodiment of the present invention, the lectin isMBL or ficolin; in a more preferred embodiment the lectin is MBL.

[0118] The term lectins may be naturally occurring lectins as well asvariants thereof, such as mutants, said variants being capable ofbinding a sugar.

[0119] In particular the invention is suitable for isolating MBL, whichwill be discussed more thoroughly in the following as an example of theinvention, and not as a limitation of the invention.

[0120] Lectins according to the present invention may comprise one ormore subunits. In particular collectins such as MBL may comprise one ormore subunits, and each subunit of a collectin is normally consisting of3 individual polypeptides. For example collectins such as MBL may bemonomers, dimers, trimers, tetramers, pentamers, hexamers, heptamers,octamers, nonamers, decamers, 11-mers, 12-mers of subunits or collectinssuch as MBL may comprise even more than 12 subunits.

[0121] Composition

[0122] The ratio R discussed herein may be calculated using any suitablemethod. In a preferred embodiment a quantitative estimate of the R valueof a in a sample might be done as follows:

[0123] The SDS-PAGE immunoblot is scanned into a TIFF file, or anothernon-compressed bitmap file. The pixel density along the sample bands aremeasured in a picture evaluation program, such as Scion Image forWindows 4.0 (Scion Corporation, freeware beta version onwww.scioncorp.com), and exported to a data evaluation program (likeExcel).

[0124] The migration corresponding to the predetermined molecular weightis settled from the migration of markers. In a preferred embodiment thepredetermined molecular weight is the molecular weight of the dimericlectin (for MBL about 200 kDa), and accordingly the migrationcorresponding to the dimeric lectin may be settled from the migration ofmarkers (for example Precision Protein Standards, BioRad). The R valueis calculated as the total pixel signal above reference migration point(for dimeric MBL 200 kDa), divided by the total pixel signal beneath thereference migration point (for dimeric MBL 200 kDa). R₀ is defined asthe ratio in the starting material, while R₁ is the ratio along thecorresponding evaluation line in the lectin sample recovered after theperformance of a method according to the present invention.

[0125] The degree of increase of the ratio R depends on the startingmaterial having ratio R₀, the starting material being the lectinpreparation, for example an MBL preparation. It is preferred that atleast 50% of the high molecular weight lectin of the composition has amolecular weight above the molecular weight for dimer lectins,preferably above the molecular weight for trimer lectins. When thelectin is MBL, it is thus preferred that at least 50% of the highmolecular weight MBL of the composition has a molecular weight above 200kDa, such as above 225 kDa, such as above 250 kDa, such as above 300kDa.

[0126] By producing an MBL composition according to the presentinvention, the MBL composition is preferably substantially free from anyimpurities naturally associated with the MBL when produced in a nativehost organism, for example impurities associated with MBL, when MBL ispurified from plasma MBL.

[0127] The method may be used for producing high mass MBL compositionsfrom any MBL source, such as any mammalian recombinant MBL or fromplasma. However, the MBL source is preferably recombinant MBL, morepreferably the MBL of the composition is human, such as MBL wherein theMBL subunit is assembled of three peptide sequences comprising thesequence as shown in SEQ ID NO:1 in PCT application WO 00/70043 or afunctional equivalent thereof.

[0128] Recombinant Production

[0129] Although the method may be applied to any lectin startingmaterial having both low and high mass lectin, such as MBL, it isparticularly suitable for producing high mass lectin, such as MBL from arecombinantly produced preparation.

[0130] Thus, the MBL preparation is preferably a recombinantpreparation, wherein the MBL preparation is obtained by

[0131] preparing a gene expression construct encoding human MBL peptideor a functional equivalent thereof,

[0132] transforming a host cell culture with the construct,

[0133] cultivating the host cell culture in a culture medium, therebyobtaining expression and secretion of the polypeptide into the culturemedium,

[0134] obtaining a preparation comprising a variety of MBL molecules

[0135] According to the invention, the sequences from the MBL gene maybe from the human MBL gene or from MBL genes of other animal species, inwhich the immune system in this respect is acting like the human immunesystem. An example of a preferred embodiment of a preparation of arecombinant MBL according to the invention is described in example 1 ofPCT application WO 00/70043 which is incorporated herein by reference.In the example the recombinant MBL is prepared by the use of anexpression vector comprising sequences from the human MBL gene.

[0136] The invention also concerns the use of expression vectorscomprising sequences which are functional derivatives of the sequencesof the human MBL gene. By said functional derivatives are meantsequences containing base pair alterations that lead to no functional oressentially no functional differences of the expression vector, and theMBL prepared in this way has a functionality comparable to the MBLprepared by the use of an expression vector comprising the unalteredsequences from the human MBL gene.

[0137] In addition to the purification method, it is preferred that thegene expression construct and the host cell also favour production ofhigher oligomers. Accordingly, the gene expression construct preferablycomprises at least one intron sequence from the human MBL gene or afunctional equivalent thereof. Furthermore, the gene expressionconstruct may comprise at least two exon sequences from the human MBLgene or a functional equivalent thereof. More preferably the geneexpression construct comprises at least three exon sequences from thehuman MBL gene or a functional equivalent thereof. When comprising morethan one exon, the exon sequences are preferably aligned as in the humanMBL gene.

[0138] Although preferred that the sequence comprises intron sequences,it may for some applications be convenient that the expression constructcomprises a cDNA sequence encoding a MBL subunit or a functionalequivalent thereof.

[0139] The invention features the use of MBL gene expression constructsrather than MBL cDNA constructs for expression of rMBL in mammalian celllines or transgenic animals to obtain recombinant MBL with structuralproperties under non-denaturing and denaturing conditions beingsubstantially similar to natural human MBL. By “recombinant human MBL”is meant human MBL which is expressed from engineered nucleic acids, andby “MBL gene expression constructs” is meant an expression vectorsuitable for expression in mammalian cell lines and containing exonsequences and at least one intron sequence from the human MBL gene orfrom MBL genes of other animal species, such as but not limited tochimpanzees and rhesus monkeys.

[0140] Preferably, the DNA sequences encode a polypeptide sequence asshown in SEQ ID NO:1 of patent application WO 00/70043 or a functionalequivalent, whereby a functional equivalent is as defined above. SEQ IDNO:1 corresponds to the MBL sequence having database accession NO:P11226. The equivalent may be obtained by a modification of the peptidesequence shown as SEQ ID NO:1, such as a sequence processing acorresponding property as the sequences mentioned in the presentinvention, but wherein one or more amino acids have been substitutedwith others. Preferably a functional equivalent contains conservativesubstitutions, i.e. where one or more amino acids are substituted by anamino acid having similar properties, such that a person skilled in theart of protein chemistry will expect the secondary and tertiarystructure of the protein to be unchanged. Amino acids suitable forconservative substitutions include those having functionally similarside chains. For example, hydrophobic residues, e.g. glycine, alanine,valine, leucine, isoleucine and methionine, may replace another suchresidue. Similarly, conservative substitutions may involve interchanginghydrophilic residues: (e.g.: arginine and lysine, glutamine andaspargine, threonine and serine), basic reduces (e.g., lysine, arginineand histidine), and/or acidic residues (e.g., aspartic acid and glutamicacid). Functional equivalents may also, or alternatively, be modified byfor example the deletion or addition of amino acids, or the chemicalmodification of amino acids, as long as the function of the polypeptideis preserved.

[0141] The isolated MBL peptide, including any functional equivalentsthereof, may in one embodiment comprise at least 80 amino acid residues,such as at least 100 amino acid residues, such as at least 150 aminoacid residues, such as at least 200 amino acid residues, for example atleast 220 amino acid residues, such as at least 250 amino acid residues.

[0142] In a preferred embodiment the expression vector is suitable forexpression in mammalian cell lines or transgenic animals which containexon sequences and at least one intron sequence from the human MBL geneor from MBL genes of other animal species, such as, but not limited to,chimpanzees and rhesus monkeys. In one embodiment the host cell cultureis cultured in a transgenic animal. By a transgenic animal in thiscontext is meant an animal which has been genetically modified tocontain and express the human MBL gene or fragments or mimics hereof.

[0143] In a preferred embodiment the expression construct of the presentinvention comprises a viral based vector, such as a DNA viral basedvector, an RNA viral based vector, or a chimeric viral based vector.Examples of DNA viruses are cytomegalovirus, Herpex Simplex virus,Epstein-Barr virus, Simian virus 40, Bovine papillomavirus,Adeno-associated virus, Adenovirus, Vaccinia virus, and Baculo virus.

[0144] In mammalian host cells, a number of viral-based expressionsystems may be used. In cases where an adenovirus is used as anexpression vector, the nucleic acid molecule of the invention may beligated to an adenovirus transcription/translation control complex, forexample, the late promoter and tripartite leader sequence. This chimericgene may then be inserted into the adenovirus genome by in vitro or invivo recombination. Insertion into a non-essential region of the viralgenome (for example, region E1 or E3) will result in a recombinant virusthat is viable and capable of expressing an MASP-3 gene product ininfected hosts (for example, see Logan and Shenk, Proc. Natl. Acad. Sci.USA 81:3655-3659, 1984). Specific initiation signals may also berequired for efficient translation of inserted nucleic acid molecules.These signals include the ATG initiation codon and adjacent sequences.In cases where an entire gene or cDNA, including its own initiationcodon and adjacent sequences, is inserted into the appropriateexpression vector, no additional translational control signals may beneeded. However, in cases where only a portion of the coding sequence isinserted, exogenous translational control signals, including, perhaps,the ATG initiation codon, must be provided. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner et al., Methodsin Enzymol. 153:516-544, 1987).

[0145] Examples of RNA virus are Semliki Forest virus, Sindbis virus,Poko virus, Rabies virus, Influenza virus, SV5, Respiratory Syncytialvirus, Venezuela equine encephalitis virus, Kunjin virus, Sendai virus,Vesicular stomatitis virus, and Retroviruses.

[0146] Examples of chimetic viruses are Adenovirus, Sindbis virus andAdenovirus—adeno-associated virus.

[0147] Regarding specific vectors, reference is made to Makrides, S. C.,“Components of vectors for Gene Transfer and Expression in MammalianCells”, which is hereby incorporated by reference.

[0148] In particular, an Epstein-Barr virus origin of replication orfunctional derivatives or mimics hereof including the pREP9 vector isused.

[0149] In one aspect the invention provides an expression constructencoding human MBL, featured by comprising one or more intron sequencesfrom the human MBL gene including functional derivatives hereof.Additionally, it contains a promoter region selected from genes of virusor eukaryotes, including mammalian and insects.

[0150] The promoter region is preferably selected to be different fromthe human MBL promoter, and preferably in order to optimize the yield ofMBL and size distribution of MBL oligomers, the promoter region isselected to function most optimally with the vector and host cells inquestion.

[0151] In a preferred embodiment the promoter region is selected from agroup comprising Rous sarcoma virus long terminal repeat promoter, andcytomegalovirus immediate-early promoter, and elongation factor-1 alphapromoter.

[0152] In another embodiment the promoter region is selected from genesof microorganisms, such as other viruses, yeasts and bacteria.

[0153] In order to obtain a greater yield of recombinant MBL, thepromoter region may comprise enhancer elements, such as the QBI SP163element of the 5′ end un-translated region of the mouse vascularendothelian growth factor gene. The construct is used for transforming ahost cell to obtain a host cell culture capable of expressing MBL. Thehost cell culture is preferably a eukaryotic host cell culture. Bytransformation of an eukaryotic cell culture is in this context meantintroduction of recombinant DNA into the cells. The expression constructused in the process is characterised by having the MBL encoding regionselected from mammalian genes including human genes and genes with bigresemblance herewith such as the genes from the chimpanzee. Theexpression construct used is furthermore featured by the promoter regionbeing selected from genes of virus or eukaryotes, including mammaliancells and cells from insects.

[0154] The process for producing recombinant MBL according to theinvention is characterised in that the host cell culture is preferablyeukaryotic, and for example a mammalian cell culture. A preferred hostcell culture is a culture of human kidney cells, and in an even morepreferred form the host cell culture is a culture of human embryonalkidney cells (HEK cells). The invention features the use of HEK 293 celllines for production of recombinant human MBL. By “HEK 293 cell lines”is meant any cell line derived from human embryonal kidney tissue suchas, but not limited to, the cell lines deposited at the American TypeCulture Collection with the accession numbers CRL-1573 and CRL-10852.

[0155] Other cells may be chicken embryo fibroblast, hamster ovarycells, baby hamster kidney cells, human cervical carcinoma cells, humanmelanoma cells, human kidney cells, human umbilical vascular endotheliumcells, human brain endothelium cells, human oral cavity tumour cells,monkey kidney cells, mouse fibroblast, mouse kidney cells, mouseconnective tissue cells, mouse oligodendritic cells, mouse macrophage,mouse fibroblast, mouse neuroblastoma cells, mouse pre-B cells, mouse Blymphoma cells, mouse plasmacytoma cells, mouse teratocacinoma cells,rat astrocytoma cells, rat mammary epithelium cells, COS, CHO, BHK, 293,VERO, HeLa, MDCK, WI38, and NIH 3T3 cells.

[0156] In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (forexample, glycosylation) and processing (for example, cleavage) ofprotein products may be important for the function of the protein.Different host cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins and geneproducts. Appropriate cell lines or host systems can be chosen to ensurethe correct modification and processing of the foreign proteinexpressed. To this end, eukaryotic host cells which possess the cellularmachinery for proper processing of the primary transcript,glycosylation, and phosphorylation of the gene product may be used. Themammalian cell types listed above are among those that could serve assuitable host cells.

[0157] The host cell culture may be cultured in any suitable culturemedium. Examples of culture medium are RPMI-1640 or DMEM supplementedwith, e.g., insulin, transferrin, selenium, and foetal bovine serum.

[0158] Purification

[0159] As discussed above the present invention allows a fast productionof MBL. The production of MBL may be conducted by the following steps:

[0160] Fermentation—culturing of MBL expressing cells

[0161] Isolation—applying the method of the invention

[0162] Accordingly, the lectin composition may be further purified byany suitable means, either prior to the isolation applying the methodsof the present invention or subsequent to the isolation according to themethod of the invention. For example the lectin composition may befurther purified by any physico-chemical isolating method, including butnot limited to filtration methods, precipitation methods,chromatography, such as ion-exchange based on charge, gel permeationbased on size, hydrophobic interaction based on hydrophobicity, oraffinity chromatography.

[0163] In addition it is possible to apply the methods according to thepresent invention more than once within one isolation procedure. Inparticular, two or more different methods according to the presentinvention may be applied in an isolation procedure, wherein the methodsmay differ by the choice of solid support, choice of sugar derivativeand/or choice of predetermined concentration of a sugar derivativecoupled to the solid support.

[0164] For example, it is possible to perform the isolation of lectinsfirst using a solid support which has coupled thereto a firstpredetermined concentration of a sugar derivative, and then performingthe isolation of lectins using a solid support which has coupled theretoa second predetermined concentration of a sugar derivative, wherein thefirst predetermined concentration is may be higher or may be lower thanthe second predetermined concentration.

[0165] In particular when isolating intermediate molecular weightlectins, a two-step isolation process using at least two different solidsupports with two different concentrations of sugar derivative may beused. In one example of a two-step isolation process the desired lectinsare obtained in the contaminant fraction, i.e. the unbound fractionhaving a molecular weight below the predetermined molecular weight inthe first step, for example any lectins below heptamers. In the secondstep the unbound fraction is applied to a different solid support havinganother cut-off point with respect to the predetermined molecularweight, for example binding any lectins above dimer molecular weight.Thereby the desired intermediate molecular weight composition oftrimers, tetramers, pentamers and hexamers is obtained when elutinglectins bound to the second solid support. It is apparent to the personskilled in the art that the two steps of course may be conducted in thereverse order to obtain the same composition.

[0166] Functionality

[0167] The functionality of the recombinant MBL composition obtainedaccording to the invention preferably resembles the functionality ofplasma or serum MBL. In the present context the functionality of MBLmeans the capability of activating the complement system as discussedabove in relation to functional equivalents. The functionality may beexpressed as the specific activity of MBL, such as units of MBL activityper ng MBL. The functionality of the recombinant MBL composition asexpressed as specific activity is preferably at least 25% of thespecific activity of MBL purified from serum, such as at least 50% ofthe specific activity of MBL purified from serum, more preferred atleast 75% of the specific activity of MBL purified from serum.

[0168] The functionality of MBL may be estimated by its capacity to forman MBL/MASP complex leading to activation of the complement system. WhenC4 is cleaved by MBL/MASP, an active thiol ester is exposed and C4becomes covalently attached to nearby nucleophilic groups. A substantialpart of the C4b will thus become attached to the coated plastic well andmay be detected by anti-C4 antibody.

[0169] A quantitative TRIFMA for MBL functional activity was constructedby 1) coating microtitre wells with 1 mg of mannan in 100 ml of buffer;2) blocking with Tween-20; 3) applying test samples, e.g. diluted MBLpreparations 4) applying MBL deficient serum (this leads to theformation of the MBL/MASP complex); alternatively the MBL and the MBLdeficient serum may be mixed before application with the microtitrewells; 5) applying purified complement factor C4 at 5 mg/ml; 6) incubatefor one hour at 37° C.; 7) applying Eu-labelled anti-C4 antibody; 8)applying enhancement solution; and 9) reading the Eu by time resolvedfluorometry. Between each step the plate is incubated at roomtemperature and washed, except between step 8 and 9.

[0170] Estimation by ELISA may be carried out similarly, e.g. byapplying biotin-labelled anti-C4 in step 7; 8) apply alkalinephosphatase-labelled avidin; 9) apply substrate; and 10) read the colourintensity. A calibration curve can be constructed using dilutions of oneselected normal plasma. In relation to the present invention thefollowing serum is an example of useful serums: plasma pool LJ 6.57 Apr.28, 1997. The functionality may be expressed as the specific activity ofMBL, such as in units of MBL activity per ng of MBL.

[0171] Another assay for determining a functional equivalent of MBL isto determine the ability to bind to receptor/receptors on cells.

[0172] The interaction of MBL with receptor/receptors on cells may beanalysed by cytofluorimetry. 1) MBL at a concentration of 50 μg/ml isincubated with 2×10⁵ cells. The binding is carried out in phosphatebuffered salt solution (PBS) containing 1% FCS and 0.1% Na-azide. 2) Fordetection of cell-bound MBL, biotinylated anti-MBL antibody is applied;3) followed by the addition of strepavidin-FITC and 4) analysis of themixture by fluorimetry.

[0173] Said diseases, disorders and/or conditions in need of treatmentwith the compounds of the invention comprise e.g. treatment ofconditions of deficiency of MBL, treatment of cancer and of infectionsin connection with immunosuppressive chemotherapy including inparticular those infections which are seen in connection with conditionsduring cancer treatment or in connection with implantation and/ortransplantation of organs. The invention also comprises treatment ofconditions in connection with recurrent miscarriage.

[0174] Thus, in particular the pharmaceutical composition may be usedfor the treatment and/or prevention of clinical conditions selected frominfections, MBL deficiency, cancer, disorders associated withchemotherapy, such as infections, diseases associated with humanimmunodeficiency virus (HIV), diseases related with congenital oracquired immunodeficiency. More particularly, chronic inflammatorydemyelinating polyneuropathy (CIDP, Multifocal motoric neuropathy,Multiple sclerosis, Myasthenia Gravis, Eaton-Lambert's syndrome, OpticusNeuritis, Epilepsy; Primary antiphosholipid syndrome; Rheumatoidarthritis, Systemic Lupus erythematosus, Systemic scleroderma,Vasculitis, Wegner's granulomatosis, Sjøgren's syndrome, Juvenilerheumatiod arthritis; Autoimmune neutropenia, Autoimmune haemolyticanaemia, Neutropenia; Crohn's disease, Colitis ulcerous, Coeliacdisease; Asthma, Septic shock syndrome, Chronic fatigue syndrome,Psoriasis, Toxic shock syndrome, Diabetes, Sinuitis, Dilatedcardiomyopathy, Endocarditis, Atherosclerosis, Primaryhypo/agammaglobulinaemia including common variable immunodeficiency,Wiskot-Aldrich syndrome and severe combined immunodefiency (SCID),Secondary hypo/agammaglobulinaemia in patients with chronic lymphaticleukaemia (CLL) and multiple myeloma, Acute and chronic idiopathicthrombocytopenic purpura (ITP), Allogenic bone marrow transplantation(BTM), Kawasaki's disease, and Guillan-Barre's syndrome.

[0175] The route of administration may be any suitable route, such asintravenously, intramusculary, subcutanously or intradermally. Also,pulmonal or topical administration is envisaged by the presentinvention.

[0176] In particular the MBL composition may be administered to preventand/or treat infections in patients having clinical symptoms associatedwith congenital or acquired MBL deficiency or being at risk ofdeveloping such symptoms. A wide variety of conditions may lead toincreased susceptibility to infections in MBL-deficient individuals,such as chemotherapy or other therapeutic cell toxic treatments, cancer,AIDS, genetic disposition, chronic infections, and neutropenia.

[0177] It appears that cancer patients treated by chemotherapy are oftensusceptible to infection due to adverse effects of the drug regime oncells of the immune system, which is the background for the use of MBLtherapy in the treatment of this condition. The observed low plasmaconcentrations of MBL (below 500 ng/ml) are indicative of an increasedsusceptibility to clinical significant infections, and the immunedefence of these patients can be reinforced by administration ofrecombinant or natural plasma-derived MBL.

[0178] The pharmaceutical composition may thus be administered for aperiod before the onset of administration of chemotherapy or the likeand during at least a part of the chemotherapy.

[0179] The MBL composition may be administered as a general “booster”before chemotherapy, or it may be administered to those only being atrisk of developing MBL deficiency. The group of patients being at riskmay be determined by measuring the MBL level before treatment and onlysubjecting those to treatment whose MBL level is below a predeterminedlevel. The limit for determining a low MBL level is evaluated to bebelow 500 ng/ml for most groups. The MBL level may be determined by timeresolved immunofluorescent assay as described in Example 9, ELISA, RIAor nephelometry.

[0180] Another indication for administering MBL is when the MBL level isbelow 50% of the normal level, such as below 300 ng/ml, or below 200ng/ml.

[0181] The MBL composition is administered in suitable dosage regimes,in particularly it is usually administered at suitable intervals, e.g.once or twice a week during chemotherapy.

[0182] Normally from 1-100 mg is administered per dosage, such as from2-10 mg, mostly from 5-10 mg per dosage. Mostly about 0.1 mg/kg bodyweight is administered.

[0183] Thus, in one aspect the invention concerns MBL, including rMBL,fragments or mimics hereof for use in the treatment of cancer and ofconditions of diseases and disorders of e.g. the immune system andreproductive system, said treatment consisting of creation,reconstitution, enhancing and/or stimulating the opsonic and/orbactericidal activity of the complement system, i.e. enhancing theability of the immune defence to recognise and kill microbial pathogens.

[0184] Furthermore, an aspect of the present invention is the use of arecombinant composition according to the present invention in akit-of-parts further comprising another medicament. In particular theother medicament may be an anti-microbial medicament, such asantibiotics.

[0185] Concerning miscarriage, it has been reported that the frequencyof low plasma levels of MBL is increased in patients with otherwise notexplained recurrent miscarriages, which is the background for loweringof the susceptibility to miscarriage by a reconstitution of the MBLlevel by administration of recombinant MBL in these cases.

[0186] As to the nature of compounds of the invention, it appears thatin its broad aspect, the present invention relates to compounds whichare able to act as opsonins, that is, able to enhance uptake bymacrophages either through direct interaction between the compound andthe macrophage or through mediating complement deposition on the targetsurface. A particular example hereof is MBL, a fragment or a mimichereof. The present invention is based upon the disclosure of asynthesis of a recombinant human MBL which appears to be closer to thestructure of the natural human MBL than achieved in the past.

[0187] The invention has now been explained and accounted for in variousaspects and in adequate details, but additionally it will be illustratedbelow by the non-limiting examples of preferred embodiments.

EXAMPLES Example 1 Purification of MBL Using an Immobilized Amino Sugar

[0188] MBL was recombinantly produced by human cell lines like a HEK293based cell line comprising an expression construct encoding MBL asdescribed in WO 00/70043, in an HyQ based media (HyClone), and MBL wassubsequently purified by affinity chromatography by using an immobilizedamino sugar.

[0189] A D-mannosamine column was prepared by coupling 1.0 g ofD-mannosamine (ICN Biomedicals, Cat. no.102252) to 13.5 mL suspendedNHS-activated Sepharose FF (Amersham Biosciences, Cat. no.17-0906-02).

[0190] Coupling conditions were exactly as described in the vendor'sinstructions for the NHS-activated gel: First, the resin was washed withice-cold 1 mM HCl. Subsequently, the resin was incubated with the aminosugar (100 mg/mL D-mannosamine, 10 mM NaH₂PO₄, pH=7.0) overnight at 4°C. Next day the resin was blocked with ethanolamine buffer (100 mM, pH7.0), and packed into a HR10/10 column. Finally, the column wasequilibrated 3 times with a weak Tris buffer (pH 7.4) and a weak acetatebuffer (pH=3.1) in an alternating pattern.

[0191] One litre of cell culture broth was added to the column, andeluted using an EDTA containing buffer.

[0192] An MBL specific immunoassay of the corresponding application,run-through, and eluting main peak reveals that MBL is almost entirelyfound in the eluting main peak (FIG. 1). MBL content is easily verifiedby a chromatographic technique, such as size exclusion chromatography onSuperose 6 (prepacked HR 10/30 from Amersham Biosciences), or MALDI-MSinvestigations after reduction.

Example 2 Purification of a Selected Size-Fraction of MBL Using anImmobilized Amino Sugar

[0193] Selected fractions of lectin oligomers may be obtained byadjusting the amino sugar ligand concentration.

[0194] A resin with relatively low ligand concentration was made bycoupling 0.1 g of D-mannosamine (Sigma, Cat. no.M4670) to 12.5 mLsuspended NHS-activated Sepharose FF (Amersham Biosciences, Cat.no.17-0906-02).

[0195] A similar resin with relatively high ligand concentration wasmade by coupling 1.0 g of D-mannosamine (Sigma, Cat. no.M4670) to 12.5mL suspended NHS-activated Sepharose FF (Amersham Biosciences, Cat.no.17-0906-02).

[0196] A ‘dummy’ column with no ligand was made by using a couplingbuffer without the amino sugar during the coupling to 12.5 mL suspendedNHS-activated Sepharose FF (Amersham Biosciences, Cat. no.17-0906-02).

[0197] In all cases, coupling conditions were exactly as described inthe vendor's instructions for the NHS-activated gel. One litre of cellculture broth (made as described in Example 1) is added to each column,and eluted using an EDTA containing buffer. An MBL specific immunoassayof the corresponding application, run-through, and eluting main peakreveals that MBL is almost entirely found in the eluting main peak fromthe column with high ligand concentration (‘high-sub’ column).

[0198] A selected high-mass fraction of MBL is found in the eluting mainpeak from the column with low ligand concentration (‘low-sub’ column),and the remaining MBL is seen in the run-through fraction. All MBL isfound in run through fraction of the ‘dummy’ column, showing that theMBL affinity is ligand specific (FIG. 2).

Example 3 Purification of a Selected Size-Fraction of MBL Using a Seriesof Columns with Immobilized Amino Sugars

[0199] Specific fractions of lectin oligomers are obtained by applying aseries of columns with different ligand concentrations.

[0200] One litre of cell culture broth (made as described in Example 1)is added to two columns in series: The first column is a column with alow amino sugar ligand concentration (a ‘low-sub’ column as described inExample 2), while the second is a column with a high amino sugar ligandconcentration (a ‘high-sub’ column as described in Example 2).

[0201] The run-through fraction of MBL from the ‘low-sub’ column iscaptured on the ‘high-sub’ column. Each column is then eluted with EDTAbuffer (50 mM, pH 7.4). The eluting fraction from the ‘low-sub’ columncontains the high mass size MBL with a mean size distribution higherthan the original application. The eluting fraction from the ‘high-sub’column contains a fraction of MBL with mass less than the ‘low sub’column eluate, but without low mass size MBL, like monomeric MBL ordimeric MBL.

Example 4 Purification of a Plasma Derived MBL Using an ImmobilizedAmino Sugar

[0202] Lectins from plasma might be purified using immobilized aminosugars.

[0203] A D-mannosamine column is prepared as described in Example 1. Onehalf litre of blood plasma is obtained from a blood bank, and coagulatedby weak heating after addition of CaCl₂. The obtained serum is added tothe column, and after a short wash protein is eluted using an EDTAcontaining buffer. An MBL specific immunoassay of the correspondingapplication, run-through, and eluting main peak reveals that MBL isalmost entirely found in the eluting main peak from the column.

[0204] MBL content is easily verified by a chromatographic technique,such as size exclusion chromatography on Superose 6 (prepacked HR 10/30from Amersham Biosciences), anion exchange chromatography on MonoQ (HR5/5 from APBiotech), or MBL ELISA (Statens Serum Institute, Copenhagen).

Example 5 Purification of MBL Using Immobilized Amino Sugars Other thanD-mannosamine

[0205] Immobilized D-mannosamine may be exchanged with any amino sugarcapable of binding the lectin after immobilization via the NH₂ group.The same effects of ligand concentration are seen.

[0206] A resin with relatively low ligand concentration was made bycoupling 0.1 g of D-glucosamine (Sigma, Cat. no.G4875) to 12.5 mLsuspended NHS-activated Sepharose FF (Amersham Biosciences, Cat.no.17-0906-02). A similar resin with relatively high ligandconcentration was made by coupling 1.0 g of D-glucosamine (Sigma, Cat.no.G4875) to 12.5 mL suspended NHS-activated Sepharose FF (AmershamBiosciences, Cat. no.17-0906-02). In both cases, coupling conditionswere exactly as described in the vendor's instructions for theNHS-activated gel.

[0207] One litre of cell culture broth (made as described in Example 1)is added to each column, and eluted using an EDTA containing buffer.

[0208] An MBL specific immunoassay of the corresponding application,run-through, and eluting main peak reveals that MBL is almost entirelyfound in the eluting main peak from the column with high ligandconcentration (‘high-sub’ column) (FIG. 3).

Example 6 Large Scale Purification of Lectins Using an Immobilized AminoSugar

[0209] The affinity chromatography is easily scaled up ten times.

[0210] A large D-glucosamine column is prepared by coupling 5.0 g ofD-glucosamine (Sigma, Cat. no.G4875) to 100 mL suspended NHS-activatedSepharose FF (Amersham Biosciences, Cat. no.17-0906-02). Couplingconditions were exactly as described in the vendor's instructions forthe NHS-activated gel. Ten litres of cell culture broth withrecombinantly produced MBL are added to the column, and eluted using anEDTA containing buffer.

[0211] An MBL specific immunoassay of the corresponding application,run-through, and eluting main peak reveals that MBL is almost entirelyfound in the eluting main peak. MBL content is easily verified bychromatographic techniques, such as size exclusion chromatography onSuperose 6 (prepacked HR 10/30 from Amersham Biosciences), anionexchange chromatography on MonoQ (Amersham Biosciences), SDS-PAGE withwesterns using Hyb131-01 (Statens Serum Institute), MBL specific ELISA's(Statens Serum Institute), amino acid composition analysis, and MALDI-MSinvestigations after reduction.

Example 7 Detection of Lectins Using Immobilized Amino Sugars

[0212] Amino sugars can be immobilized to microtiter wells and beapplied for detection of lectins.

[0213] D-glucosamine is coupled to microtiter wells (Cyanuric chlorideactivated CovaLink NH₂ from Life Technologies) by incubating the wellswith 1.0 μg amino sugar per well overnight, as described by vendor.Dilutions of MBL containing samples are added to the wells and incubatedfor 3 hours at room temperature. Subsequently a detection antibody likeHyb131-01 (Statens Serum Institute) is applied for 3 hours at roomtemperature, followed by incubation with a HRP- or AP-labelled secondaryantibody (anti-IgG, Dako) for 1 hour. After addition of the enzymesubstrate, the amount of MBL is estimated in an ELISA reader.

1. A method for isolating at least one lectin from a preparationcomprising a variety of lectin molecules, comprising establishing asolid support having coupled thereto a predetermined concentration of asugar derivative, applying the preparation to said solid support,allowing the lectins to bind to the sugar derivative coupled to thesolid support, washing the solid support, with a washing liquid removingunbound contaminants, and optionally recovering said lectin(s) from thesolid support.
 2. The method according to claim 1, wherein the sugarderivative is an amino sugar.
 3. The method according to claim 2,wherein the amino sugar is selected from the group consisting ofgalactosamine, mannosamine, glucosamine, allosamine, altrosamine,ribosamine, arabinosamine, gulosamine, idosamine, fucosamine,talosamine, xylosamine, lyxosamine, sorbosamine, tagatosamine,psicosamine and fructosamine.
 4. The method according to claim 2,wherein the amino sugar is selected from the group consisting ofD-mannosamine, D-glucosamine, D-allosamine, D-altrosamine, D-ribosamine,D-arabinosamine, L-gulosamine, L-idosamine, L-galactosamine,L-fucosamine, L-talosamine, L-xylosamine and L-lyxosamine.
 5. The methodaccording to claim 2, wherein the amino sugar is selected from the groupconsisting of D-mannosamine and D-glucosamine.
 6. The method accordingto any of the preceding claims, wherein the lectin is recovered fromsaid solid support.
 7. The method according to claim 6, wherein thelectin is recovered in substantially pure form.
 8. The method accordingto any of the preceding claims, wherein the solid support is selectedfrom chromatography columns, chromatographic resins, filters, plasticsurfaces, glass surfaces and magnetic particles.
 9. The method accordingto claim 8, wherein the column is selected from N-hydroxy succinimideactivated resins, Cyanogen bromide activated resins, Epoxy activatedresins, ECH Sepharose 4B from Amersham Biosciences and ActivatedCH-Sepharose 4B from Amersham Biosciences.
 10. The method according toclaim 8, wherein the plastic surface is an activated microtiter plate.11. The method according to claim 1, wherein the lectin is MBL.
 12. Themethod according to any of the preceding claims, wherein the washingliquid comprises one or more components selected from the groupconsisting of sugars capable of binding to the lectin, sugar derivativescapable of binding to the lectin and divalent metal ion chelatingagents.
 13. The method according to claim 12, wherein the sugar isselected from the group consisting of mannose, mannosamine,N-acetylmannosamine, glucose, glucosamine, N-acetylglucosamine,fructose, fructosamine, N-acetylfructosamine, galactose, galactosamine,N-acetylgalactosamine, saccharose and maltose.
 14. A method forincreasing the ratio R of a composition comprising a variety of lectinmolecules, wherein R is the ratio of the concentration of lectinmolecules having a high molecular weight above a predetermined molecularweight to the concentration of lectin molecules having a low molecularweight below or equal to the predetermined molecular weight, said methodcomprising obtaining a lectin preparation comprising low molecularweight lectin and high molecular weight lectin, said preparation havingthe ratio R=R₀, applying the preparation to an isolation method asdefined in any of claims 1 to 13, obtaining a composition comprising therecovered lectin molecules.
 15. A method for producing a compositioncomprising a variety of lectin molecules, wherein substantially all ofsaid lectin molecules having a molecular weight above a predeterminedmolecular weight for dimer lectins, said method comprising obtaining alectin preparation comprising lectin molecules having a high molecularweight above the predetermined molecular and lectin molecules having alow molecular weight below or equal to the predetermined molecularweight, said preparation having the ratio R=R₀, wherein R is the ratioof the concentration of lectin molecules having a high molecular weightabove the predetermined molecular weight to the concentration of lectinmolecules having a low molecular weight below or equal to thepredetermined molecular weight, applying the preparation to an isolationmethod as defined in any claims 1 to 13, obtaining a compositioncomprising the recovered lectin molecules.
 16. A method for separating acomposition comprising a variety of lectin molecules, whereinsubstantially all of said lectin molecules have a high molecular weightabove a predetermined molecular weight, from a lectin preparationcomprising lectin molecules having a high molecular weight above thepredetermined molecular weight and lectin molecules having a lowmolecular weight below or equal to the molecular weight for dimerlectins, said preparation having the ratio R=R₀, wherein R is the ratioof the concentration of lectin molecules having a high molecular weightabove the predetermined molecular weight to the concentration of lectinmolecules having a low molecular weight below or equal to thepredetermined molecular weight, said method comprising obtaining saidlectin preparation, applying the preparation for an isolation method asdefined in any of claims 1 to 13, obtaining a composition comprising therecovered lectin molecules.
 17. A method for producing a compositioncomprising a variety of lectin molecules, wherein substantially all ofsaid lectin molecules having a low molecular weight below or equal to apredetermined molecular weight, said method comprising obtaining alectin preparation comprising lectin molecules having a high molecularweight above the predetermined molecular weight and lectin moleculeshaving a low molecular weight below or equal to the predeterminedmolecular weight, said preparation having the ratio R=R₀, wherein R isthe ratio of the concentration of lectin molecules having a highmolecular weight above the predetermined molecular weight to theconcentration of lectin molecules having a low molecular weight below orequal to the predetermined molecular weight, applying the preparation toan isolation method as defined in any of claims 1 to 13, obtaining acomposition comprising the unbound lectin molecules of the washingliquid.
 18. A method of increasing the ratio R of a preparationcomprising a variety of lectin molecules, wherein R is the ratio of theconcentration of lectins having an intermediate molecular mass to theconcentration of lectin molecules having a high molecular mass and a lowmolecular mass, wherein intermediate molecular mass is a molecularweight below a first predetermined molecular weight and above a secondpredetermined molecular weight, high molecular mass is a molecular massabove or equal to the first predetermined molecular weight and lowmolecular mass is a molecular mass below or equal to the secondpredetermined molecular weight, said method comprising the steps of a)obtaining a lectin preparation comprising low molecular mass lectin,intermediate molecular mass lectin and high molecular mass lectin, saidpreparation having the ratio R=R₀; and b) applying the preparation to anisolation method as defined in any of claims 1 and 13; and c) obtaininga composition comprising the recovered lectin molecules; and d)optionally repeating step b)
 19. The method according to any of thepreceding claims 14 and 18, wherein the lectin is mannose-binding lectin(MBL).
 20. The method according to claim 18, wherein at least 50% of thehigh molecular weight MBL of the composition has a molecular weightabove 200 kDa, such as above 225 kDa, such as above 250 kDa, such asabove 300 kDa.
 21. The method according to claim 18, wherein the lowmolecular weight MBL comprises MBL having a molecular weight below 200kDa.
 22. The method according to any of the preceding claims, whereinthe MBL preparation is a recombinant MBL preparation.
 23. The methodaccording to claim 22, wherein the MBL preparation is obtained bypreparing a gene expression construct encoding human MBL peptide or afunctional equivalent thereof, transforming a host cell culture with theconstruct, cultivating the host cell culture in a culture medium,thereby obtaining expression and secretion of the polypeptide into theculture medium, obtaining a preparation comprising a variety of MBLmolecules
 24. The process according to claim 23, wherein the geneexpression construct comprises at least one intron sequence from thehuman MBL gene or a functional equivalent thereof.
 25. The processaccording to claim 24, wherein the gene expression construct comprisesat least two exon sequences from the human MBL gene or a functionalequivalent thereof.
 26. The process according to claim 23, wherein thegene expression construct comprises a cDNA sequence encoding a MBLsubunit or a functional equivalent thereof.
 27. The process according toclaim 23, wherein the host cell culture is cultured in vitro.
 28. Theprocess according to claim 23, wherein the host cell culture is aeukaryotic host cell culture.
 29. The process according to claim 23,wherein the host cell culture is a mammalian host cell culture.
 30. Asolid support, wherein said solid support is having covalently coupledthereto a predetermined concentration of at least one amino sugar. 31.The solid support according to claim 30, wherein the amino sugar isselected from the group consisting of galactosamine, fucosamine,mannosamine, glucosamine, allosamine, altrosamine, ribosamine,arabinosamine, gulosamine, idosamine, galactosamine, talosamine,xylosamine, lyxosamine, sorbosamine, tagatosamine, psicosamine andfructosamine.
 32. The solid support according to claim 30, wherein thesolid support is selected from chromatography columns, chromatographicresins, filters, plastic surfaces, glass surfaces and magneticparticles.
 33. The solid support according to claim 30, wherein thecolumn is selected from N-hydroxy succinimide activated resins, Cyanogenbromide activated resins, Epoxy activated resins, ECH Sepharose 4B fromAPBiotech and Activated CH-Sepharose 4B from AP-Biotech.
 34. A methodfor isolating at least one lectin from a preparation comprising avariety of lectin molecules, comprising establishing a solid supportbeing derivatized with at least one amino sugar, applying thepreparation to said solid support, allowing the lectins to bind to theamino sugar coupled to the solid support, washing the solid support witha washing liquid removing unbound contaminants, and optionallyrecovering said lectin(s) from the solid support.
 35. The methodaccording to claim 34, wherein the method is as defined in any of claims3-13.